The ability of T cells to mediate in vivo anti-tumor effects in humans is well documented, but reliably harnessing this activity to provide therapeutic benefit has proven difficult. Many obstacles have been identified, including selecting an appropriate tumor antigen to target, generating T cells with sufficient avidity in adequate numbers to recognize and eliminate tumor cells expressing the selected antigen, maintaining the response in the context of persistent antigenic stimulation, and overcoming inhibitory and tolerizing pathways resulting from the tumor/self-antigens being targeted. Adoptive T cell therapy, with autologous tumor-reactive T cells generated and expanded ex vivo and then infused into the patient to establish a large effector response, has provided means to bypass many limitations for in vivo immunization to tumors antigens. However, substantive impediments to reproducible therapeutic success remain. Strategies such as genetically modifying T cells prior to transfer to impart the desired specificity and requisite avidity for target recognition or to enhance effector activity, and/or disrupting regulatory pathways operative in the tumor-bearing host, offer opportunities to create therapies with improved efficacy. Determining which modifications to pursue and how and in what settings to deploy such techniques and reagents would benefit from testing in informative mouse models. [unreadable] [unreadable] This application represents a continuation of our efforts using murine models to address issues relevant to clinical translation of adoptive T cell therapy. The studies focus on two major experimental areas: retroviral mediated transfer of genes into T cells as a means to provide specificity and enhance function in the potentially hostile environment of a progressive tumor; and transgenic, knockout, and knock-in mouse models developed by us and collaborators in which tumor-reactive T cell responses can be readily monitored and manipulated, and are targeting candidate tumor antigens also detected in normal tissues, which represents a common clinical setting and obstacle. Molecular strategies that impart T cells with gain of function to improve T cell activity and avidity for tumor targets or loss of function to disrupt intracellular inhibitory pathways that limit the responsiveness of T cells will be pursued, as well as methods to ablate CD4+ TREG cells and/or the activity of TGF[unreadable] or IL10 on the tumor-reactive T cells. The ultimate goals are to identify strategies with potential for translation to the treatment of human malignancies. The specific aims are to: 1) Determine if TCR genes genetically modified to improve expression, affinity, and/or signaling impart T cells with enhanced function and improved efficacy in tumor therapy; 2) Determine if CD8 T cell therapeutic activity can be augmented by reducing the expression/function of cellular inhibitory proteins that mitigate T cell activation and responses; 3) Determine if tumor-reactive T cells can be generated in hosts to a potentially tolerogenic candidate tumor antigen expressed in both normal prostate tissue and spontaneously developing prostate cancer, and if CD8 T cells bearing a high affinity TCR specific for the self-antigen can therapeutically target the tumor. [unreadable] [unreadable] [unreadable]